An early step in acute pancreatitis (AP) is premature zymogen (protease) activation. We hypothesize that rapid acidification of the acinar cell cytosol has a central role in modulating this response. In addition, published data from our laboratory suggests that zymogen activation is mediated via a proton-pumping vacuolar ATPase (vATPase) and that generation of a low-pH compartment within the pancreatic acinar cell is critical for this activation. Given that clinical studies have shown a correlation between metabolic acidosis and AP, we further hypothesize that pH effects on zymogen activation are dependent on vATPase activity. Transient cytosolic acidification is observed upon stimulation of isolated acinar cells with physiologic concentrations of the cholecystokinin (CCK) orthologue cerulein (10-10 M). However, induction of experimental AP with 10-7 M cerulein shows a more prominent and prolonged cytosolic acidification. In our proposal we have manipulated cytosolic pH using either medium acidification or direct intracellular acidosis (with sodium propionate) and observed that either mechanism of acidification sensitized the acinar cell to supraphysiologic cerulein-induced zymogen activation. Further, we have shown using vATPase inhibitors bafilomycin and concanamycin, that vATPase mediates zymogen activation with extracellular acidification. We propose that cytosolic acidification may have a critical role in premature zymogen activation by sensitizing acinar cells to secretagogue stimulation. In forms of acute pancreatitis associated with metabolic acidosis or decreased pancreatic blood flow, this is likely to be an important pathologic mechanism. Lay Statement: Acute Pancreatitis (AP) has an incidence of about 80,000 cases per year in the United States with an overall mortality of 5%, yet the underlying biological mechanism of this disease is largely unknown. Clinical observations suggest that acidosis predisposes to AP. Our planned studies will examine the hypothesis that acidosis sensitizes the pancreas to the development of AP. One implication of our findings is that reducing acidosis in specific clinical situations could reduce the risk and/or severity of AP. [unreadable] [unreadable] [unreadable]